Highly biocompatible dual thermogelling chitosan/glucosamine salt composition

ABSTRACT

The present disclosure relates to a chitosan solution neutralized with amino-sugar carbonate buffering solution or amino-sugar phosphate buffering solution or phosphorylated aminosugar buffering solution. The resulting thermogelling chitosan composition is highly biocompatible, isotonic and has the ability to rapidly turn into gel upon heating to the body temperature. It provides a novel chitosan-based composition to suitable for drug delivery, cell delivery and repair or regeneration of tissues and organs as well as other clinical treatment.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 13/218,516, filed Aug. 26, 2011, which claims priority from of U.S.Provisional Application Ser. No. 61/377,592, filed Aug. 27, 2010, andU.S. Provisional Application Ser. No. 61/444,646, filed Feb. 18, 2011,the specification of which are hereby incorporated by reference.

TECHNICAL FIELD

The present description relates to chitosan solution neutralized withamino-sugar carbonate buffering solution, amino-sugar phosphatebuffering solution or phosphorylated amino-sugar buffering solution.

BACKGROUND ART

Hydrogels are continuously gaining increased attention as biomaterialsfor biomedical applications, such as tissue engineering and therapeuticsdelivery. Furthermore, in situ forming hydrogels or those exhibiting thespecific ability of increasing their viscosity with temperature, alsocalled thermosensitive, are preferred over preformed hydrogels, sincecells and bioactive compounds, such as drugs, may be easily mixed withthe precursor solutions prior to gelation to give homogeneously loadedgels. In addition, in situ gelation facilitates the application andallows for minimally invasive surgery and for adequately fill complexshaped lesion cavities.

Chitosan is an amino polysaccharide obtained by partial to substantialalkaline N-deacetylation of chitin also namedpoly(N-acetyl-D-glucosamine), which is a naturally occurring biopolymerfound in exoskeleton of crustaceans, such as shrimp, crab and lobstershells. Chitosan contains free amine (—NH₂) groups and may becharacterized by the proportion of N-acetyl-D-glucosamine units andD-glucosamine units, which is expressed as the degree of deacetylation(DDA) of the fully acetylated polymer chitin. The properties ofchitosan, such as the solubility and the viscosity, are influenced bythe degree of deacetylation (DDA), which represents the percentage ofdeacetylated monomers, and the molecular weight (Mw).

Chitosan has been proposed in various formulations, alone and with othercomponents, to stimulate repair of dermal, corneal and hard tissues in anumber of reports (U.S. Pat. Nos. 4,572,906; 4,956,350; 5,894,070;5,902,798; 6,124,273; and WO 98/22114). The properties of chitosan thatare most commonly cited as beneficial for the wound repair process areits biodegradability, adhesiveness, prevention of dehydration and as abarrier to bacterial invasion. The interesting haemostatic potential ofchitosan has also led to its direct application to reduce bleeding atgrafts and wound sites (U.S. Pat. No. 4,532,134). Some studies claimthat the haemostatic activity of chitosan derives solely from itsability to agglutinate red blood cells while others believe itspolycationic amine character can activate platelets to release thrombinand initiate the classical coagulation cascade thus leading to its useas a haemostatic in combination with fibrinogen and purified autologousplatelets (U.S. Pat. No. 5,773,033).

One technical difficulty that chitosan often presents is a lowsolubility at physiological pH and ionic strength, thereby limiting itsuse in a solution state. Thus typically, dissolution of chitosan isachieved via the protonation of amine groups in acidic aqueous solutionshaving a pH ranging from 3.0 to 5.6. Such chitosan solutions remainsoluble up to a pH near 6.2 where neutralisation of the amine groupsreduces interchain electrostatic repulsion and allows attractive forcesof hydrogen bonding, hydrophobic and van der Waals interactions to causepolymer precipitation at a pH near 6.3 to 6.4. Admixing apolyol-phosphate dibasic salt (i.e. glycerol-phosphate) to an aqueoussolution of chitosan can increase the pH of the solution while avoidingprecipitation. In the presence of these particular salts, chitosansolutions of substantial concentration (0.5-3%) and high molecularweight (>several hundred kDa) remain liquid, at low or room temperature,for a long period of time with a pH in a physiologically acceptableneutral region between 6.8 and 7.2. This aspect facilitates the mixingof chitosan with cells in a manner that maintains their viability. Anadditional important property is that such chitosan/polyol-phosphate(C/PP) aqueous solutions solidify or gel when heated to an appropriatetemperature that allows the mixed chitosan/cell solutions to be injectedinto body sites where, for example cartilage nodules can be formed insubcutaneous spaces.

Chitosan is thus recognized as a biodegradable, biocompatible,antibacterial and haemostatic biopolymer that is able to promote woundhealing, drug absorption, and tissue reconstruction. Due to the abovementioned intrinsic properties, chitosan also has been widely exploredin numerous cosmetic and pharmaceutical applications. Therefore,considering the great potential of chitosan, there is a continuous needto improve the properties of known thermosensitive chitosan hydrogelswhich are still considered as very promising for a wider range ofbiomedical applications.

U.S. Pat. No. 6,344,488, discloses a pH-depend temperature controlledchitosan composition prepared by neutralizing a commercial chitosanhaving a deacetylation degree ranging from 70 to 95% with mono-phosphatedibasic salts of polyols or sugars, phosphorylated polyols orphosphorylated sugars, exemplified in particular with β-glycerophosphate(β-GP). Because of its unique properties, the thermogelling chitosan-GPsystem has raised significant biomedical interest. However, highconcentration of β-GP was required, particularly for chitosan having DDAbetween 70 and 85%, in order to achieve fast gelation at bodytemperature and to avoid rapid elimination of the hydrogel after itsadministration (Chemte et al., 2000, Biomaterials, 21: 2155-2161; andChemte et al., 2001, Carbohydrate Polymers, 46: 39-47). This resultedinto very high osmolarity, more than twice of that of physiologicalextracellular fluid (Crompton et al., 2007, Biomaterials, 28: 441-449;and Hoemann et al., 2005, Osteoarthritis Cartilage, 13: 318-329).Ideally, the hydrogel should be isotonic with the extracellular fluid;and its osmolarity should be around 300 mOsm. The osmolarity is a veryimportant factor regulating biocompatibility of the hydrogel with cellseither in vitro or in vivo.

Further, in an attempt to improve the gelation properties of chitosan-GPsystem, particularly for isotonic compositions, U.S. patent applicationpublication No. 2009/0202430 proposed the addition of glyoxal aschemical crosslinker. In another description, particular composition ofchitosan-GP system has been combined with blood in the attempt toimprove and stabilize blood clots (U.S. Pat. No. 7,148,209 and U.S.patent application publication No. 2010/0178355).

U.S. patent applications Nos. 2009/0270514 and 2010/0113618 describedthe preparation of thermogelling chitosan solutions by using, instead ofβ-GP, either (NH₄)₂HPO₄ solution or NaOH solution respectively. However,the use of ammonium phosphate salts or all the salts derived fromorganic bases as disclosed in U.S. patent applications No. 2009/0270514may be harmful or damageable to cells and living tissues, even if theyare at a concentration which normally leads to isotonic thermogellingchitosan solutions. U.S. patent applications No. 2010/0113618 wasrestricted to reacetylated chitosan having a degree of deacetylation(DDA) ranging from 30 to 60%. Moreover, the NaOH solution is beforehandadded with high concentration of 1,3-propanediol, an organic reagentwhich can be potentially toxic to cells and living tissues. Despite theslight improvement provided by the use polyoses or polyols instead1,3-propanediol, as disclosed in U.S. patent applications No.2009/0004230, the toxicity problem remain unsolved, so the system cannot be a suitable matrix for cells, sensitive proteins or livingtissues.

It is also well known that a solution of bicarbonate salt as NaHCO₃, aweak base, can be used to increase the pH of chitosan solution in thevicinity of 6.5 without causing any precipitation, but the resultingsolution is unable to turn into homogeneous hydrogel in temperaturerange between 0 and 50° C. In fact, a pseudo-gelation can be observed,occurring at the surface of the solution caused by the release of CO₂,as has been reported by recent study (Liu et al., 2011, Int. J. Pharm.,414: 6-15). In such a case, to achieve gelation of the whole sample, itis necessary to disturb the solution and bring ungelled solution to thesurface from the bottom of the sample. This leads to non homogeneoushydrogel.

Thus, there is still a need to be provided with an improvedthermogelling chitosan solution having better biocompatibilityproperties, that is not toxic to cells and living tissue.

SUMMARY

In accordance with the present description there is now provided abiocompatible thermogelling composition comprising chitosan; and abuffering solution;

In an embodiment, the composition is liquid at a pH between 6.5 and 7.6and at a temperature between about 15° C. and about 22° C., and forms agel when heated up to a temperature range between about 25 and about 60°C.

The composition disclosed herein can also form gels when cooled down toa temperature between about 8° C. and about 1° C., a temperature around4° C.

It is also provided herein a method of preparing a thermogellingcomposition of chitosan, comprising the steps of dissolving chitosan inan acidic solution to obtain an aqueous solution of chitosan; andadmixing a buffering solution to the aqueous chitosan solution, toobtain the thermogelling composition of chitosan as described herein.

In an embodiment, the temperature is maintained between 15° C. and 22°C. (room temperature range) during the preparation.

In accordance with the present disclosure, it is also provided a methodfor delivering a material or compound to a subject in need thereof,comprising the steps of admixing the thermogelling composition describedherein with the material or compound; and administering the admixedcomposition and material and/or compound to the subject.

It is also provided a method of treating, repairing, regenerating,replacing or substituting a tissue or organ within a mammalian or humanbody comprising the step of administering the composition describedherein.

It is also provided the use of the composition described herein fortreating, repairing, regenerating, replacing or substituting a tissue ororgan within a mammalian or human body.

In a preferred embodiment, the buffering solution is an amino-sugarcarbonate solution, an amino-sugar phosphate solution or aphosphorylated amino-sugar solution.

In another embodiment, the buffering solution is a glucosamine carbonatesolution, a glucosamine phosphate solution or a glucosamine-6-phosphatesolution.

The thermogelling composition described herein can have a pH between 6.7and 7.2.

In another embodiment, the thermogelling composition is in a liquidstate at temperature between 15° C. and 22° C.

In a further embodiment, the thermogelling composition turns into gelwhen heated up to a temperature of 37° C. or cooled down to atemperature around 4° C.

The concentration of chitosan can range from 0.1% to 5.0% or from 1.0%to about 3.0%; the concentration of glucosamine carbonate, glucosaminephosphate or glucosamine-6-phosphate can range from 0.002M to 0.100M.

In a preferred embodiment, the ratio of chitosan to glucosaminecarbonate, of chitosan to glucosamine phosphate and/or the ratio ofchitosan to glucosamine-6-phosphate is approximately between 1 and 3.There is a direct relationship between this ratio and the pH of thethermogelling composition and the gelation temperatures.

In another embodiment, the chitosan has a degree of deacetylation (DDA)ranging between 70% and 100% and a molecular weight (Mw) ranging from 50kDa to 1000 kDa; preferably a DDA of 80% to 99%, and a Mw of 200 kDa to500 kDa.

In another embodiment, the thermogelling composition can furthercomprise at least one material or compound, such as for example but notlimited to cells, stem cells, peptides, growth factors, human blood,platelet-rich plasma, nucleotides, drugs and/or imaging agents.

In another embodiment, the osmolarity of said composition is between 270mOsmol/kg and 340 mOsmol/kg.

In another embodiment, the composition is injected to a tissue defect ina patient, then gelled in the tissue defect.

In an alternate embodiment, the composition is pregelled before beinginjected in a tissue defect in a patient.

In a further embodiment, the composition is administered in order totreat, repair, regenerate, replace or substitute, either totally orpartially, a tissue or organ within a mammalian or human body.

In another embodiment, the composition is injected intrarticular totreat or improve body joint functions, or to repair cartilage defects.

In another embodiment, the material or compound is calcium phosphateparticles at a concentration comprising between 1.0% and 40.0%.

The calcium phosphate particles can be biphasic calcium phosphates,tetra-calcium phosphates, tri-calcium phosphates, hydroxyapatite,di-calcium phosphates, mono-calcium phosphates, amorphous calciumphosphates, octa-calcium phosphate, fluorinated calcium phosphate,strontied calcium phosphate, or a mixture thereof.

Particularly, the biphasic calcium phosphate particles are sized from 50to 1000 microns.

Further particularly, the biphasic calcium phosphates comprise from 20%to 85% of tri-calcium phosphate; and from 80% to 15% of hydroxyapatite.

In a further embodiment, the composition is injected and administered asan homogeneous gel.

Particularly, the homogeneous gel has a setting time from 1 minute to 30minutes, and turns into a composite solid scaffold.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings.

FIG. 1 illustrates the evolution of the elastic modulus and viscousmodulus with the temperature of a thermogelling composition having a pHvalue around 6.7 (chitosan DDA=98%) as described herein.

FIG. 2 shows a thermogelling composition as described herein undergoingdual thermogelation. This typical image illustrates dual thermogelationobtained for chitosan solution (DDA=80% or 98%) neutralized either withglucosamine carbonate buffering solution or with glucosamine phosphatebuffering solution.

DETAILED DESCRIPTION

It is provided an aqueous chitosan solution neutralized with amino-sugarcarbonate buffering solution, with amino-sugar phosphate bufferingsolution or with phosphorylated amino-sugar buffering solution. Theresulting thermogelling chitosan composition is highly biocompatible,isotonic and has the ability to rapidly turn into gel upon heating tothe body temperature. In a preferred embodiment, the chitosan solutionis neutralized either with an amino-sugar carbonate buffering solution,with a glucosamine phosphate buffering solution or with aglucosamine-6-phosphate dibasic buffering solution.

The present description discloses the preparation of thermogellingchitosan hydrogels neutralized with a buffering solution of glucosaminecarbonate, glucosamine phosphate and/or of glucosamine-6-phosphate.Glucosamine carbonate and glucosamine phosphates are salts where thecation is none other than the positively charged glucosamine, which isthe repeating unit in chitosan itself. Either glucosamine orglucosamine-6-phosphate are abundantly found in human tissue and joints,and improve the biocompatibility and bioactivity of the thermogellingchitosan solutions.

The thermogelling chitosan solutions described herein are neutral andhighly biocompatible, and can be used in a wide array of biomedicalapplications as injectable hydrogels for controlled and prolongeddelivery of drugs, proteins and growth factors, injectable fillers,injectable composites, as tissue adhesive and wound dressing materialsand as scaffolds for tissue engineering applications.

It is described herein the preparation of a chitosan solution, havingphysiological pH, able to undergo thermogelation upon heating up toaround body temperature. In one aspect, the thermogelling orthermosetting chitosan solution is prepared by admixing appropriateamounts of glucosamine carbonate solution or of glucosamine phosphatesolution to chitosan solution at room temperature, preferably between 15and 22° C., under vigorous stirring. The resulting solutions, even at pHbetween 6.7 and 7.2, have been found to remain liquid at roomtemperature and turn into hydrogels when heated up to 37° C. or above.The time required for gelling to occur has been found to mainly dependon the temperature and the pH of the final solution, which in turndepend on the amount of glucosamine hydrogen phosphate solution called“buffering solution” and the concentration of chitosan solution. In oneaspect, the final pH of an efficient thermogelling chitosan solutionshould be at least about 6.7. In a separate embodiment, thethermogelling chitosan solutions can also form hydrogels upon cooling toa temperature between 8 and 1° C.

The thermogelling chitosan composition described herein is highlybiocompatible with cells, sensitive proteins and living tissues, asglucosamine carbonate or glucosamine phosphate, are used as thebuffering solutions, the virtues of glucosamine being conserved.Glucosamine is an amino-sugar naturally synthesized from glucose andglutamine, an amino acid. It is abundant in human joints where it is akey precursor for the biochemical synthesis of various compoundsincluding glycolipids, glycoproteins, glycosaminoglycans, hyaluronateand proteoglycans. All those compounds are present in cartilage andother joint components where they fulfill important roles for jointresilience and lubrication.

With age, the body gradually loses its ability to convert glucose andglutamine into glucosamine, due to lower levels of the converting enzymeglucosamine synthetase. This gradual decrease has been suggested to beone of the main factors contributing to degenerative joint diseases suchas osteoarthritis (OA). Clinical group studies and claims by patientssupport the fact that a daily supplement of glucosamine over a period oftime can have beneficial effects for OA patients. Apparently,glucosamine might act to improve cartilage resilience by stimulating invivo the biosynthesis of glucosaminoglycan.

Exoskeleton of crustaceans, such shrimp, crab and lobster shells areusually the source of commercial glucosamine, which is obtained by thebreak down or the degradation of chitosan to the monomer unit.

Clinical studies and claims by patients support the fact that a dailysupplement of glucosamine over a period of time can have beneficialeffects for OA patients. From a safety viewpoint, human studies haveconsistently reported that the administration of glucosamine did notaffect the plasma levels of glucose or insulin, insulin sensitivity orglucose oxidation (Scroggie et al., 2003, Archives of Internal Medicine,163: 1587-1590; Pouwels et al., 2001, J. Clin. Endocrinol. Metab., 86:2099-2103; and Monauni et al., 2000, Diabetes, 49: 926-935). Thisindicates that glucosamine had no significant effect on blood glucosemetabolism even in patients with type 2 diabetes mellitus.

Anderson and coworkers reviewed the clinical trial data recorded formore than 3000 patients, and stated that the oral administration ofglucosamine was moderately to highly effective in treatingosteoarthritis pain, and had no adverse effects on blood, urine or fecalparameters (Anderson et al., 2005, Food and Chemical Toxicology, 43:187-201). Furthermore, the review summarizes results about very highdoses of glucosamine administered orally to rats, mice, rabbits, dogsand horses, as reported in nearly 20 animal studies. The LD₅₀ wasestimated to exceed 5000 mg/kg for rats and 8000 mg/kg for mice andrabbits. The investigation also showed that the ingestion of glucosamineat high doses, ranging from 300 to 2149 mg/kg of body weight, have noeffect on blood glucose levels in rats, rabbits or dogs. Moreover, infifty-four outpatients with gonarthrosis, a double-blind clinical testwas conducted with the aim of evaluating the efficacy and tolerance ofintra-articular Glucosamine in comparison with a 0.9% NaCl placebo. Eachpatient had one intra-articular injection per week for five consecutiveweeks. Pain, active and passive mobility of the joint, swelling, andgeneralized and local intolerance symptoms were recorded beforebeginning the treatment, and four weeks after the last injection.Glucosamine reduced pain to a significantly greater extent than didplacebo, and resulted in significantly more pain-free patients. Theangle of joint flexion substantially increased after glucosaminetreatment. Active mobility increased with both treatments, with a morefavourable trend after glucosamine administration. Knee swelling did notdecrease significantly after glucosamine, whereas it worsened (althoughno significantly) after placebo. There were no local or generalintolerance symptoms during and after treatment.

Glucosamine administration was able to accelerate the recovery ofarthrosic patients, with no resulting side effects, and to partiallyrestore articular function. In addition, the clinical recovery did notfade after treatment ended, but lasted for the following month, atleast. Glucosamine therapy therefore was shown to deserve a selectedplace in the management of osteoarthrosis (Vajaradul et al., 1981, ClinTher., 3:336-343). Chitosan has been registered to GRAS (GenerallyRecognized As Safe). Chitosan composition and materials have beenextensively analyzed in vitro as well as in vivo, both in animals andhumans. In vitro, chitosan compositions have been tested with variouscell lines, including Caco-2 cells, HT29-H, CCRF-CEM (humanlymphoblastic leukaemia), and L132 (human embryonic lung cells), MCF7and COS7 cells (Kean et al., 2010, Advanced Drug Delivery Reviews, 62:3-11; Richardson et al., 1999, Int. J. Pharm., 178: 231-243; Schipper etal., 1996, Pharm. Res., 13: 1686-1692; Schipper et al., 1999, Eur. J.Pharm. Sci., 8: 335-343; and Zhang, et al., 2008, Biomaterials, 29:1233-1241).

In vivo, chitosan compositions and materials have been tested in variousanimal models and through several administration routes. Chitosan hasbeen safely studied in mouse models (immunogenicity), rat models, guineapig models, and rabbit models (subacute toxicity). No “significant toxiceffects” of chitosan were noted in acute toxicity tests in mice, no eyeor skin irritation in rabbits and guinea pigs respectively. In the samestudy it was also concluded that chitosan was not pyrogenic. Exposure ofrat nasal mucosa to chitosan solutions at 0.5% (w/v) over 1 h caused nosignificant changes in mucosal cell morphology compared to control. Frommost studies reported it appears that chitosan shows minimal toxiceffects and this justifies its selection as a safe material in drugdelivery. Chitosan/b-Glycerophosphate systems have been investigated invitro, in vivo in animal models and in humans, and have shown a safe andnon-toxic profile (Hirano et al., 1991, Agric. Biol. Chem., 55:2623-2625; Ono et al., 2000, J. Biomed. Mater. Res., 49: 289-295; Azadet al., 2004, J. Biomed. Mater. Res. B Appl. Biomater., 69: 216-222;Ishihara et al., 2001, Wound Repair Regen., 9: 513-52; and Illum et al.,1994, Pharm. Res., 11: 1186-1189).

In humans, a phase 2 clinical trial involving the percutaneous injectionof chitosan-¹⁶⁶holmium complex, for the treatment of hepatocellularcarcinoma, on patients with poor surgical prospects, reported safe andefficacious results. The effects of chitosan have been investigated oneighty patients with renal failure undergoing long-term stablehaemodialysis treatment. The patients were tested after a controltreatment period of 1 week. Half were fed 30 chitosan tablets (45 mgchitosan/tablet) three times a day. Ingestion of chitosan effectivelyreduced total serum cholesterol levels (from 10.14+/−4.40 to 5.82+/−2.19mM) and increased serum haemoglobin levels (from 58.2+/−12.1 to 68+/−9.0g L-1). During the treatment period, no clinically problematic symptomswere observed. The results suggest that chitosan might be an effectivetreatment for renal failure patients, although the mechanism of theeffect should be investigated further.

Chitosan was also administrated intranasally to deliver morphine inpatients following orthopaedic surgery, and was shown to offer a safeand less invasive alternative to intra venous (IV) morphine. An clinicaland pharmacokinetic study for a drug delivery system (DDS) ofgentamycin-loaded chitosan bar were carried out with the purpose toevaluate its efficacy and giving further data for its clinicalapplications. Eighteen (18) cases of chronic osteomyelitis were treatedby surgical necrectomy with implantation of gentamycin-load chitosan barin the prepared bone cavity. All of the 18 cases were followed up for24.8 months (in a range of 6-34 months) 16 patients received initialcure and without any recurrence. So, it could be concluded that thegentamycin-loaded chitosan DDS was a simple and effective method for thetreatment of chronic osteomylitis without the necessity to carry out asecond operation to remove the drug carrier.

In China, on 12 patients, chitosan was observed to safely prevent orreduce elbow adhesion after elbow arthrolysis. It was investigated againin humans to prevent knee adhesion following patella operation (Kim etal., 2006, Clin. Cancer Res., 12: 543-548; Jing et al., 1997, J PharmPharmacol., 49(7): 721-723; Stoker et al., 2008, Pain Med., 9: 3-12; andChen et al., 1998, Chinese Journal of Reparative and ReconstructiveSurgery, 12: 355-358).

Several clinical trials involving chitosan compositions or materials fordrug delivery or medical implant purposes are ongoing (recruiting) orterminated in the United States. Chitosan materials are, or have been,clinically studied in patients for the management of difficultspontaneous epistaxis and to evaluate its healing effect on nasalmucosa, to investigate the safety and efficacy of hemostasis of thedressing for use in dental surgical procedures, to test a chitosan padafter diagnostic percutaneous coronary angiography as an adjunct tomanual compression to better control vascular access site bleeding andreduce time-to-hemostasis, to investigate a chitosan composition as asafe, effective debridement of chronic wounds in the operating room andinpatient ward settings and to minimize bacterial re-colonization ofwounds, to investigate the therapeutic benefits of using a chitosancomposition for the wound repair of diabetic neuropathic foot ulcers, tocompare the efficacy of a chitosan composition versus conventionaltreatment in the treatment of Diabetic Neuropathic Foot Ulcer, toinvestigate a new chitosan derivative for reducing the symptomsassociated with Dry Eye Syndroma, and to investigate whether thetreatment of damaged cartilage in the knee with a chitosan compositionwill increase the amount and quality of cartilage repair tissue whencompared with microfracture alone. Moreover, chitosan materials are, orhave been, clinically studied in patients to determine if chitosan, ashort-chained chitosan with a molecular weight of 40 kDa, is safe andeffective in lowering LDL-cholesterol levels in patients with mild tomoderately elevated cholesterol levels (drug), and to compare safety andimmunogenicity of two dosage levels of Norwalk VLP Vaccine with chitosanadjuvant/excipients.

It is also disclosed herein the preparation of highly biocompatiblethermogelling solutions of chitosan by using naturally occurringglucosamine-6-phosphate in solution or in solid form.Glucosamine-6-phosphate is the intermediate product in the pathwayleading to the natural biosynthesis of glucosamine, recognized as thebiochemical precursor of all nitrogen-containing sugars (Roseman, 2001,J. Biol. Chem., 276: 41527-41542), which are important constituents ofglycoproteins and oligosaccharides involved in biological recognition.Specifically, glucosamine-6-phosphate is synthesized fromfructose-6-phosphate and glutamine (Ghosh et al., 1960, J. Biol. Chem.,235: 1265-1273) as the first step of hexosamine biosynthesis pathway.The end-product of this pathway is uridine diphosphateN-acethylglucosamine or UDP-GlcNAc, a nucleotide sugar used then formaking glycosaminiglycans, proteoglycans and glycolipids.

It is conceived herein that any phosphorylated amino-sugar can be usedas described hereinabove. Furthermore, contrary to U.S. Pat. No.6,344,488, the content of which is incorporated herein by reference,which teaches the use of monophosphate of polyols and sugars(phosphorylated polyols and sugars), any person having ordinary skill inthe art will make the distinction that the present disclosure isdirected to the use of amino-sugars which is different from sugarsand/or polyols.

Sugar refers to a number of carbohydrates, such as monosaccharides,disaccharides, or oligosaccharides. Monosaccharides are also called“simple sugars,” having the formula C_(n)H_(2n)O_(n), where n is between3 and 7. Glucose, which has the molecular formula C₆H₁₂O₆, is the mostimportant monosaccharide. The carbohydrates are really justpolyhydroxyaldehydes, called aldoses, or polyhydroxyketones, calledketoses, while polyols are simply alcohols containing multiple hydroxylgroups. Chitosan compositions described in the art, such as in U.S. Pat.No. 6,344,488, encompassed sugars being monosaccharide such asmono-phosphate di-basic sugars, mono-sulfate sugars and mono-carboxylicsugars.

An amino-sugar as encompassed herein is a sugar where a hydroxyl groupis substituted with an amine group. Derivatives of amine-containingsugars, such as N-acetylglucosamine, while not formally containing anamine, are also considered amino-sugars.

Phosphorylation is the chemical addition of a phosphate (PO₄) group to aprotein, sugar or other organic molecule. As used herein,glucosamine-6-phosphate refers to glucosamine phosphorylated on carbon6.

As used herein, “amino-sugar carbonate solution” or “amino-sugarphosphate solution” refers to a solution containing positively chargedamino-sugar (+NH₃-sugar) among counter-ions needed to balance negativelycharged CO₃ ²⁻ and PO₄ ³⁻, so that the total charge is zero.

As used herein, “phosphorylated amino-sugar solution” refers to asolution where the negatively charged ion is the amino-sugar-phosphate(amino-sugar-O—PO₃ ²⁻).

The term “gelating temperature” is intended to mean any temperatureranging from about 25° C. to about 70° C., preferably between 37° C. toabout 60° C., and more preferably at about the physiological temperatureor 37° C.

The expression “in situ gelation” refers herein to the formation of gelsfollowing injection of the liquid chitosan solution as taught hereinwithin specific sites of mammalian or human environments, e.g. anytissues (muscles, bone, ligaments, cartilages) and organs. Gelation insitu allows complete and precise filling of tissue defects or bodycavities. The gelation of the chitosan mixture is induced by thephysiological temperature.

A chitosan gel as taught herein is an ideal material for drug deliverysystem. Such an in situ gel-like forming vehicle, wherein a solidparticulate or water-soluble additive is incorporated prior to thegelation, can be administrated topically, directly to the body site tobe treated or diagnosed. Anti-bacterial, antifungal, steroidal ornon-steroidal anti-inflammatory, anti-cancer, anti-fibrosis, anti-viral,anti-glucoma, miotic and anti-cholinergies, anti-psychotic,antihistaminic and decongestant, anesthesic and anti-parasitic agentsmay be incorporated within the composition and gel. In a similarfashion, polypeptides or non-living pharmaceutical agents may beincorporated within the composition or gel for restorative,reconstructive or regenerative purposes.

The present disclosure will be more readily understood by referring tothe following examples which are given to illustrate embodiments ratherthan to limit its scope.

EXAMPLE I Preparation of a Mixture of Chitosan-Buffering Solution

1. Preparation of Chitosan Solution

Chitosan solution (2.00% w/v) was prepared by dissolving medical gradechitosan, having medium molecular weight, in aqueous solution of HCl.The ratio of HCl compared to the chitosan amino group (NH₂), referred asthe degree of protonation of chitosan in solution, was maintained at70%. The solution was sterilized using an autoclave for 30 minutes at121° C. After cooling, the water lost caused by the autoclave processwas compensated by adding sterile water under controlled asepticenvironment. The solution was then aseptically filtered through a metalfrit, partitioned in 5.0 mL aliquots and stored at 4° C. An extraaliquot of about 3 mL was used to measure the pH of the chitosansolution. The characteristics of 100 mL solutions prepared usingchitosan having DDA of about 80% and 98% are summarized in Table 1.

TABLE 1 Characteristics of chitosan solution (100 mL) Chitosan DDA (%) m(g) H₂O (mL) HCl, 1M (mL) pH 80 2.0566 93.20 6.80 5.51 98 2.0586 91.268.74 5.502. Preparation of Buffering Solutions

Buffering solution of glucosamine-carbonate was obtained byco-dissolving simultaneously glucosamine hydrochloride and sodiumcarbonate in water, while the buffering solution of glucosaminephosphate was prepared by dissolving simultaneously glucosaminehydrochloride and tribasic potassium phosphate. The amounts of salt usedfor the preparation of 50 mL of each buffering solution are summarizedin Table 2. Generally, the pH of the buffering solution is maintainedbetween 7.60 and 8.00 for glucosamine carbonate and between 8.10 and8.50 for glucosamine phosphate.

For long-term stability, the buffering solutions of glucosaminecarbonate and glucosamine phosphate should be stored at very lowtemperature, below −20° C., preferentially −80° C. This can prevent orstop a probable Maillard reaction-like, which has been suspected to becausing the degradation, revealed by the browning coloration, of thebuffering solutions when stored at temperatures above 0° C. Technically,to solve this problem, the buffering solutions glucosamine carbonate andglucosamine phosphate can be prepared at the time of use by mixing avolume of doubly concentrated solution of glucosamine-chloride with asame volume of doubly concentrated solution of carbonate salts or bymixing a volume of doubly concentrated solution of glucosamine with asame volume of doubly concentrated solution of phosphate salts,respectively. These solutions, namely, glucosamine-chloride solution,carbonate solution or phosphate solution, prepared separately, can bestored at 4° C. for at least more than 6 months. At this temperature,degradation does not occur in acidic aqueous solutions of glucosaminehydrochloride, while aqueous solutions of carbonate or phosphate saltsare pretty stable.

TABLE 2 Amounts of salt used for buffering solution Buffering solution(50 mL) Glucosamine Glucosamine Components carbonate phosphateGlucosamine-HCl (g) 8.9808 8.9858 Na₂CO₃ (g) 2.9704 — K₃PO₄ (g) — 5.3562pH 7.68 8.393. Preparation of Thermogelling Solutions Using Glucosamine CarbonateI. Chitosan DDA=80%

The thermogelling solution was prepared by vigorously mixing 5.00 mL ofchitosan solution with 0.56 mL of glucosamine carbonate bufferingsolution, while maintaining the temperature around 15° C. The resultingsolution having a pH of about 6.82 was then poured in a test tube andincubated at 37° C., where it gelled within approximately 10 minutes.

In a second experiment, 5.00 mL of chitosan solution was mixed undervigorous stirring with 0.50 mL of glucosamine-cabonate solution, whilemaintaining the temperature around 15° C. The resulting solution havinga pH value of about 6.75, gelled within 20 minutes at 45° C.

II. Chitosan DDA=98%

The thermogelling solution was prepared by vigorously mixing 5.00 mL ofchitosan solution with 0.50 mL of glucosamine carbonate bufferingsolution, while maintaining the temperature around 15° C. The resultingsolution having a pH of about 6.8 was then poured in a test tube andincubated at 37° C., where it gelled within approximately 1 minute.

In a second experiment, 5.0 mL of chitosan solution was mixed undervigorous stirring with 0.40 mL of glucosamine-cabonate solution, whilemaintaining the temperature around 15° C. The resulting solution havinga pH value of about 6.7, gelled within 20 minutes at 45° C. Thetemperature dependence of elastic modulus (G′) and viscous modulus (G″)of the latter solution is shown in FIG. 1.

4. Preparation of Thermogelling Solutions Using Glucosamine Phosphate

I. Chitosan DDA=80%

The thermogelling solution was prepared by mixing, under vigorousstirring, 5.00 mL of chitosan solution with 0.60 mL of glucosaminephosphate solution, while maintaining the temperature around 15° C. Theresulting solution having a pH of about 7.02 was then poured in a testtube and incubated at 37° C., where it gelled within about 7 minutes.

In a second experiment, 5.00 mL of chitosan solution was mixed undervigorous stirring with 0.50 mL of glucosamine phosphate solution, whilemaintaining the temperature around 15° C. The resulting solution havinga pH value of about 6.81, gelled within 30 minutes at 45° C.

However, thermogelling composition disclosed herein cannot be obtainednor by using glucosamine hydrochloride solution nor by using freeglucosamine solution. As the pH of 3.11 measured for a 0.55M glucosaminehydrochloride solution is much lower than that of chitosan solution, thepH of the mixture does not exceed a value of 5.50. Such mixtures remainliquid in the whole range of temperature, from 0 to 80° C. In contrastthe use of free glucosamine solution, with pH of 7.71 and 8.03,increases the pH of the mixture, but a substantial precipitation ofchitosan occurred as soon as a pH value between 6.2 and 6.4 was reached.

Also, a solution of Na₂CO₃ cannot be used to prepare the thermogellingcomposition disclosed herein. When added to chitosan solution, therelatively strong alkalinity of such a carbonate solution (0.373M), pHabout 11.5, causes instantaneous precipitation of chitosan. Then acids,including but not limited to organic acids such as glutamic acid andpyruvic acid have been used to soften the alkalinity of carbonatesolution and thus provide buffering solution for thermogellingcomposition disclosed herein. However, these buffering solutions havebeen found to be less effective than glucosamine carbonate and bufferingsolution. Table 3 shows the amounts needed for the preparation ofglutamic-carbonate solutions with pH values of 7.65 and 7.85. Thecompositions resulting from the mixing of 5.00 mL of chitosan solution(DDA=98%) with 0.50 mL of solution 1, and with 0.50 mL of solution 2,had respectively a pH value of 6.31 and 6.56.

TABLE 3 Amounts needed for the preparation of glutamic-carbonatesolutions Glutamic-carbonate Glutamic acid Na₂CO₃ (50 mL) (g) (g) pHSolution 1 4.5675 3.2875 7.65 Solution 2 6.6525 4.9965 7.85

EXAMPLE II Preparation of a Thermogelling Solution of Chitosan UsingGlucosamine-6-Phosphate

Chitosan solution (˜2.0% w/v) was prepared as described above in ExampleI. The thermogelling solution was prepared by mixing 5.0 mL ofrefrigerated chitosan solution with 0.5 mL of refrigeratedglucosamine-6-phosphate disodium salt solution (1M) in ice bath (˜4°C.), and under vigorous stirring. The resulting solution having a pH ofabout 7.0 was then taken out of the ice bath and placed at 37° C., whereit gelled within 15 minutes.

EXAMPLE III Therapeutic Procedures with Dual Thermogelling Composition

The composition disclosed herein can be used for minimally-invasivetherapeutic procedures, particularly on musculo-skeletal tissues such asarticular cartilage, fibrocartilage and bone to name only a few of them.The composition described herein is particularly suited for treatingarticular cartilage injuries, and has been clinically applied inpatients suffering articular cartilage defects. This composition hasbeen applied by orthopaedic specialists, under a clinical protocol andunder the Special Access Program (SAP) from Health Canada, to treatarticular cartilage defects in knee joints of patients suffering kneecartilage injuries, knee joint pain and reduced joint functionalities.

A total of 9 patients, aged from 18 to 70 year old, had intact kneeligament structures and suffered from one-compartment symptomaticcartilage lesions, with the cartilage lesions being investigated by themagnetic resonance imaging (MRI), have been treated in Canada. Allpatients were treated arthroscopically with the debridement of thenon-adherent articular cartilage and the composition was administeredarthroscopically to fill and cover the cartilage defects. The cartilagedefects treated with the composition were up to 3 cm×3 cm in surfacearea. The composition acts primarily to fill articular cartilage defectsand resurfaces the injured cartilage surfaces in the joint. Thecomposition administered in patient' knees has proven to be safe,non-toxic, and easy to prepare and to administer. With a follow-up of 8to 9 months post-op, all patients treated with the composition showedclear positive clinical outcomes beginning at 3 to 6 months post-op.,such positive clinical outcomes consisting primarily in significantlyreduced knee joint pain and in improved knee joint functionality andoverall patient activity level. Clinical evaluation was performed usingWOMAC type scoring and questionnaire. The composition proposed fortreating articular cartilage injuries can be used for treating cartilagedefects in body joints for knee and other joints, especially in the hipand ankle.

The treatment with the composition described herein is performed duringthe course of a knee arthroscopy. It is done along with a wash anddebridement, and can be associated with a bone marrow stimulatingtechnique (microfracture). The composition can be applied directly ontothe Articular Cartilage Injury.

The composition described herein is prepared easily and rapidly duringthe course of a knee arthroscopy procedure. Furthermore, since it can beadministered as an injectable, it is advantageously very easilyadministered through an arthroscopy and it does not significantlylengthen the duration of arthroscopic procedures.

The treatment of Articular Cartilage Injury with the compositiondescribed herein reduced knee joint pain and improved knee jointfunctions, thus providing enhanced joint functionalities and overallactivity level to the treated patients. These beneficial effects shouldoccur as soon as at 3 months post-arthroscopy. This treatment canpostpone more aggressive and costly prosthetic treatments of ArticularCartilage Injury.

While the description has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the disclosure following, in general, theprinciples of the disclosure and including such departures from thepresent disclosure as come within known or customary practice within theart to which the disclosure pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A method of treating, repairing, regenerating,replacing or substituting a tissue or organ within a mammalian or humanbody comprising the step of administering a thermogelling compositioncomprising: a) a chitosan solution; and b) a buffering solutionconsisting of an amino-sugar carbonate solution, an amino-sugarphosphate solution or a phosphorylated amino-sugar solution; wherein thecomposition, being liquid at a pH between 6.5 and 7.6, is biocompatible,isotonic and turns into gel when heated up to a temperature rangebetween about 25° C. and about 60° C., and cooled down to a temperaturerange between about 8° C. and about 1° C.
 2. The method of claim 1,wherein said buffering solution is a glucosamine carbonate solution, aglucosamine phosphate solution or a glucosamine-6-phosphate solution. 3.The method of claim 1, wherein the composition is injected then gelledin the mammalian or human body.
 4. The method of claim 1, wherein saidcomposition is pregelled before being injected in mammalian or humanbody.
 5. The method of claim 1, wherein the tissue or organ comprisesarticular cartilage, fibrocartilage, meniscus, intervertebral discs,bone tissues, muscular tissues, nerve and spinal cord soft-tissues, skinor dermal tissues.
 6. The method of claim 1, wherein the composition isinjected intrarticular to treat or improve body joint functions, or torepair cartilage defects.
 7. The method of claim 1, wherein saidcomposition is prepared by: a) dissolving chitosan in an acidic solutionto obtain the aqueous solution of chitosan; and b) admixing thebuffering solution to said chitosan solution.
 8. The method of claim 7,wherein the chitosan is dissolved at a temperature between 15 and 22° C.9. The method of claim 1, wherein said composition is in liquid statehaving a pH between 6.7 and 7.2.
 10. The method of claim 1, wherein saidcomposition forms a gel when heated up to 37° C.
 11. The method of claim1, wherein the concentration of chitosan ranges from 0.1% to 5.0%. 12.The method of claim 2, wherein the concentration of glucosaminecarbonate, glucosamine phosphate or glucosamine-6-phosphate ranges from0.002M to 0.100M.
 13. The method of claim 1, wherein the concentrationof chitosan ranges from 1.0% to about 3.0%.
 14. The method of claim 2,wherein the ratio of chitosan to glucosamine carbonate, glucosaminephosphate or glucosamine-6-phosphate is between 1 and
 3. 15. The methodof claim 1, wherein said chitosan has a degree of deacetylation (DDA)ranging between 70% and 100% and a molecular weight (Mw) ranging from 50kDa to 1000 kDa.
 16. The method of claim 1, wherein said chitosan has adegree of deacetylation (DDA) of 80% to 99%, and a Mw of 200 kDa to 500kDa.
 17. The method of claim 1, wherein the osmolarity of saidcomposition is between 270 mOsmol/kg and 340 mOsmol/kg.
 18. The methodof claim 1, further comprising at least one material or compoundselected from the group consisting of cells, stem cells, peptides,growth factors, human blood, platelet-rich plasma, nucleotides, bone,bone-derived materials, calcium phosphates, calcium carbonates,bioglasses, ceramics, drugs and imaging agents.
 19. The method of claim18, wherein said calcium phosphate particles are biphasic calciumphosphates, tetra-calcium phosphates, tri-calcium phosphates,hydroxyapatite, di-calcium phosphates, mono-calcium phosphates,amorphous calcium phosphates, octa-calcium phosphate, fluorinatedcalcium phosphate, strontied calcium phosphate, or a mixture thereof.